Regulatory Peptides, 31 (1990) 41-52 Elsevier
41
REGPEP 00964
Metabolic clearance rates of oxyntomodulin and glucagon in the rat: contribution of the kidney A. Kervran ~.t, M. Dubrasquet 1,,, p. Blache ~, J. Martinez ~ and D. B ataille I Centre CNRS-INSERM de Pharmacologie-Endocrinologie, Montpellier and 21NSERM U. 10, Hrpital Bichat, Paris (France) (Received 10 January 1990; revised version received 16 July 1990; accepted 18 July 1990)
Key words: Half-life; Primed-continuous infusion technique; Nephrectomy; Hyperglycemic effect
Summary The half-life (t,/:) and metabolic clearance rate (MCR) of exogenous natural porcine oxyntomodulin (porcine OXM) and the synthetic analog of rat oxyntomodulin, [NIe27]-OXM (rat OXM), were compared with that of glucagon in control, shamoperated and acutely nephrectomized rats using the primed-continuous infusion technique. The half-disappearance times for porcine OXM (8.2 + 0.5 min) and rat OXM (6.4 + 0.5 min) were 3-fold slower than that ofglucagon (1.9 + 0.1 min). Acute bilateral nephrectomy significantly prolonged the half-disappearance time of rat OXM (8.2 + 0.7 min) and glucagon (3.6 + 0.4 min) compared with that of sham-operated animals (6.5 + 0.8 min and 2.5 + 0.2 min, respectively). The mean MCRs were similar for porcine and rat OXM (11.3 + 0.7 and 11.9 + 0.5 ml- kg- 1. min - 1) but were 3 times lower than that measured with glucagon (36 + 5 ml. kg- 1. min- 1). Bilateral nephrectomy reduced the MCR of OXM and glucagon by 38 Yo and 34Yo, respectively. No significant increase in C-terminal glucagon immunoreactivity was noticed during infusion of either porcine or rat OXM, measured directly in plasma, with a specific C-terminal glucagon antiserum or after HPLC. In the course of the glucagon infusion, blood glucose was increased 2-fold, while the same dose of porcine OXM or of rat OXM induced only a small increase over the values in phosphate buffer-infused rats. 10 times higher doses of rat OXM were necessary to obtain a similar hyperglycemic effect. These results indicate that: (1) the metabolism of OXM is 3-fold slower than that of * Deceased in 1990. Correspondence: A. Kervran, Centre CNRS-INSERM de Pharmacologie-Endocrinologie, 34094 Montpellier Cedex 5, France. 0167-0115/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
42 glucagon, (2) renal clearance contributed close to 35 ~o of the overall metabolic plasma extraction for OXM and glucagon and (3) OXM, although effective at a higher dose, when compared with glucagon, displays a hyperglycemic effect probably through the glucagon receptors.
Introduction
Oxyntomodulin (OXM) is a glucagon-containing peptide, initially isolated from porcine jejuno-ileum [1], which contains the sequence of glucagon, extended at its C-terminal end by a basic octapeptide [2]. The primary structure of human and rat OXM, deduced from the nucleotide sequence of their cDNAs are identical [3,4] and differs from that of porcine OXM by a single amino acid (Arg vs. Lys) in position 33. The distribution of OXM has been determined in the human and rat bowel [5,6] and in the rat central nervous system [7]. In rat plasma, the OXM concentration increased 2-fold 2 h after a solid meal [6]. The basic octapeptide which differentiates OXM from glucagon, confers biological activities which are quantitatively different: in vivo, OXM is 15-20 times more potent than glucagon in inhibiting pentagastrin-induced gastric acid secretion in the rat [8]; it is also one order of magnitude more potent than glucagon in inhibiting exocrine pancreatic secretion [9]. In vitro, binding sites in oxyntic gland prepared from rat gastric mucosa [ 10] had affinities 10 times higher for OXM than for glucagon. In contrast, OXM is 10 times less effective than glucagon for the production of cAMP in rat liver membranes [2]. Differences in the plasma half-life of peptides existing in different forms and bearing the same biological activity (as well as their potential interconversion from one form to another) may alter substantially their relative potencies in in vivo experiments performed on a molar-dose basis. Indeed, this was the case for somatostatin-14 and -28 [11]. Therefore, our study was designed: (1) to compare the disappearance kinetics of natural porcine OXM and synthetic analog of rat OXM to that of glucagon, (2) to determine whether the metabolism of OXM results in a significant conversion to glucagon and (3) to investigate the contribution of the kidney in the handling of these peptides.
Materials and Methods
Peptide infusion procedure and samples Male Wistar rats (250-280 g) fed ad libitum, were anesthetized by an intraperitoneal injection of pentobarbital (30 mg/kg body weight). Peptide challenging was carried out 30 min later using a primed-continuous infusion of 1.8 nmol/kg/45 min, in isotonic phosphate buffer (pH 7.4), through the saphenous vein: the prime dose (0.45 nmol/kg) was administered over 2 rain at a flow rate of 0.5 ml/min and the continuous infusion, over a period of 43 min, at a flow rate of 70/~l/min. This dosage was chosen because, in pilot experiments, it was found to induce a steady state of peptide concentration
43 within the first 15 rain of infusion. In a set of experiments, rats were bilaterally nephrectomized or sham-operated 30 min before peptide challenge. Blood samples of 0.5 ml were taken from a carotid catheter at 0, 15, 30 and 45 min throughout infusion and, at the end of infusion, at 1, 2.5, 5, 7.5, 10 and 20 min. Blood samples were collected in chilled tubes containing antiprotease (500 KIU/ml aprotinin, Biosys, Compirgne, France) and EDTA (1 mg/ml), centrifuged at 4 °C and stored at 20 ° C until assay. -
Peptides and chemicals Highly purified porcine OXM, used for infusion and column calibration, was obtained as previously described [1,2]. The synthetic analog of rat OXM [NleZT]-OXM) was obtained by solid-phase synthesis, achieved in our laboratory [12]. Rat glicentin was partially purified on a Bio-Gel P 10 column (Bio-Rad, Richmond, CA) and by reversedphase HPLC as previously described [6]. Highly purified porcine pancreatic glucagon was purchased from the Novo Research Institute (Bagsvaerd, Denmark). All solvents and reagents were of analytical grade. Reversed-phase HPLC Glucagon-containing peptides were quantitatively extracted from 5 ml plasma samples on Sep-Pak cartridges (Waters Associates, Millipore, Velizy, France) as previously described [6]. Eluates were separated at room temperature by reversed-phase HPLC on two #Bondapak C~8 columns (i.d. 0.39 x 30 cm, Waters) in series and a C18 precolumn (Waters), using a Waters chromatograph composed of two model 6000 A pumps, and a model 720 solvent programmer, a Rheodyne (model 7010) sample injector (Touzart et Matignon, Paris, France), fitted with an 18-ml loop. Reversed-phase chromatography were performed, as reported elsewhere [6] with a 45-min linear gradient of 20-50~o acetonitrile (SDS, Peypin, France) in aqueous solvent consisting of 1~o trifluoroacetic acid (Fluka, Buchs, Switzerland) buffered to pH 2.5 by diethylamine (J. T. Baker Chemicals, Deventer, The Netherlands), at a flow rate of 1.5 ml/min. Fractions were collected every 0.3 min. Chemical and immunological analyses Blood glucose concentration was measured using a glucose-oxidase method (Biochemica test combination, Boehringer, Mannheim, F.R.G.). Glucagon and glucagon-like immunoreactivity (GLI) were determined by RIAs using the C-terminal glucagon antiserum GAN and the centrally-directed glucagon antiserum GOL, as previously reported [6]. GOL antiserum displayed a full cross-reactivity with glucagon, OXM and partially purified rat glicentin and GAN antiserum showed a 2~o cross-reaction with OXM [6]. Mono 12SI-glucagon [ 13] was used as tracer and the separation of free from bound peptides was performed by adsorption on dextran-coated charcoal. Peptides bearing the C-terminal octapeptide (glicentin and OXM) were measured with an OXM/glicentin C-terminal RIA using FAN antiserum as previously reported [ 14]. In brief, the C-terminal nonadecapeptide (OXM 19-37), which fully cross-reacted with OXM and partially purified rat glicentin, and mono [ 125I-Tyr]-OXM 19-37 were used as standard and as tracer, respectively. Separation of free from bound peptides
44 was obtained by adsorption on dextran-coated charcoal. The sensitivity of FAN antiserum was 2 fmol/tube and the intra- and inter-assay reproducibilities at this concentration were 13 + 2~o (n = 16) and 23 + 3~o (n = 6), respectively.
Calculations Glucagon and OXM data, obtained from infusion studies, were analyzed according to the plateau principle [ 15,16]. Plasma steady state concentrations of glucagon or OXM were taken as the mean of the three values obtained at 15, 30 and 45 min. At the end of peptide infusion, the increment of glucagon and GLI concentrations, above the preinfusion levels, were expressed as percent of the mean plateau value, plotted on a logarithmic scale versus time and computed to yield the slope from which the tl/2 was determined. The metabolic clearance rate (MCR), expressed in ml. kg- 1. min - ~ was calculated by dividing the infused dose (pmol • kg- ~• min- 1) by the plateau increment in plasma peptide (pmol- ml- 1). The apparent volume of distribution (in ml. k g - 1) was obtained by dividing the MCR by the slope of the regression line for disappearance. Linear regression analysis was determined by the least-squares method. All values shown represent the mean + S.E.M. Differences between means was evaluated by paired or unpaired Student's t-test, as required. In this study we assumed that endogenous production of glucagon or of OXM remained constant during the exogenous infusion experiments.This assumption is indirectly supported by the steady level of glucagon, as determined by GAN antiserum, during OXM infusion (Fig. lb and c).
Results
The time-courses of the mean plasma concentrations ofglucagon and GLI during the intravenous primed-continuous peptide infusion were measured by the C-terminal glucagon antiserum (GAN) and the centrally-directed glucagon antiserum (GOL), respectively, and are shown Fig. 1. The preinfusion values of glucagon and GLI, expressed in fmol/ml of plasma glucagon equivalent, were (n = 24) 34 + 2 and 105 + 4, respectively. Plateau values were observed between 15 and 45 min after the start of the infusion of 1.8 nmol/kg of the peptides. The coefficients of variation at 15-45'min, during glucagon infusion (Fig. la), porcine OXM infusion (Fig. lb), and rat OXM infusion(Fig, lc),were6 + 1 ~ , 8 + 3~o and 10 + l~o,respectively. Theplateau values, during porcine or rat OXM infusions (Table I), were about 2.5-fold higher than that observed during glucagon infusion and they were significantly increased in nephrectomized animals (P < 0.05). During the course of infusions of porcine and rat OXM, the plateau values for glucagon, as measured with GAN antiserum, were 83 + 9 fmol/ml and 70 + 9 fmol/ml, respectively, and were significantly higher than the preinfusion level (P < 0.001). This increment, over baseline values, corresponded to 1-1.5 ~o of the plasma GLI concentration, as determined with GOL antiserum and was in good agreement with the 2~o cross-reactivity of GAN antiserum with OXM (see Materials and Methods). Thus,
45 .
a = GOL antiserum • GAN ~mfise~m
~
2'
r. 0
o
1~ T
3b
i m e
-
~
4~
60
0
~
3~ T i m e
(rain)
4~
6~
(min)
4
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g2 0 OlD
E
r. o
1~
ab
4;
T i m e
(rain)
6b
Fig. 1. Plasma concentrations of C-terminal ( m ) and central (r-q) glucagon-like immunoreactivity during and after 45 min intravenous primed-continuous infusion of 1.8 nmol/kg of: (a) glucagon (n = 8), (b) porcine O X M (n = 4) and (c)rat O X M (n = 12). Symbols and bars are m e a n s _+ S.E.M.
changes in G L I concentrations during infusion of porcine or of rat O X M can be attributed to an increase in these peptides and not to a stimulatory effect on glucagon secretion. When the peptide infusion was discontinued, circulating glucagon and G L I disappeared within the 20-min period studied, according to bi-exponential curves which are indicated on a semilogarithmic scale in Fig. 2. Examination of the decay regression
46 TABLE I Comparison of plasma half-lives (t~a), metabolic clearance rate (MCR) and apparent distribution volume (Vd) for glucagon, natural porcine oxyntomodulin (pOXM) and synthetic rat oxyntomodulin (rOXM) in control, sham-operated and nephrectomized rats Animals
Peptides
n
Plateau value (fmol/ml)
t~a (min)
MCR (ml. k g - i. min - ])
Vd (ml. kg - ~)
Control
Glucagon pOXM rOXM Glucagon rOXM Glucagon rOXM
8 4 12 5 5 5 5
1302 3669 3532 1715 3214 2095 5312
1.9 8.2 6.4 2.5 6.5 3.6 8.2
36.4 11.3 11.9 29.2 13.0 19.4 8.0
101 135 112 106 122 100 95
Sham-operated Nephrectomized
+ 104 _+ 206 + 149 + 53 + 64 _+ 153 + 257
+ 0.1 + 0.5 + 0.5 + 0.2 _+ 0.4 _+ 0.4 + 0.5
+ + + + + + +
4.5 0.7 0.5 1.0 0.3 1.0 0.2
+ 16 _+ 13 + 12 + 9 + 18 + 13 + 8
Values shown are the mean + S.E.M.
lines revealed that the disappearance rate of glucagon was much faster than that of porcine or rat OXM. The calculated values for plasma half-lives, MCR and apparent volume of distribution from the rapid decay regression lines, obtained with GOL antiserum, are provided in Table I. The t, n for porcine and rat OXM, as measured with GOL were not significantly different (P > 0.05) for the fast (Table I) and the slow (28.2 + 5.4 min vs. 23.8 + 3.5 min, respectively) regression lines. The t,/2 for glucagon 100
~2 10
1'0
1'5
Time (min) Fig. 2. Disappearance rates o f p o r c i n e O X M ( , ) , r a t O X M (F], O ) and glucagon ( A , Z~) from the plasma, as measured with G O L antiserum ( m , F], A ) , G A N antiserum ( A ) and F A N antiserum ( 0 ) . Peptide concentrations are expressed as percent of the mean 45 min plateau value. Results are m e a n s _+ S.E.M.
47
and rat OXM, were not statistically different whatever the region specific antisera used (Fig. 2). However, a significant difference was seen for the slow component of the decay regression line of rat OXM, when measured with FAN antiserum (12.8 + 1.6 min, P < 0.05). Infusion of glucagon in sham-operated rats elicited a significant increase of the plateau value (P < 0.01) and plasma t,/~ (P < 0.02), as compared to the control rats. Such an effect was not observed in sham-operated rats infused with rat OXM. Acute bilateral nephrectomy significantly prolonged the plasma half-lives of glucagon and rat OXM (P < 0.05) and decreased their MCR (P < 0.001) by 34% and 38~o, respectively, as compared to the sham-operated values. The reversed-phase HPLC profile of plasma samples (Fig. 3), taken 45 min after the start of rat OXM infusion (1.8 nmol/kg/45 min), revealed an increase in a single major peak of activity, when compared with a control plasma elution profile, that eluted in the position of the pure rat OXM peptide. Two small peaks were also seen on the elution profiles, corresponding to glicentin (retention time 30.5 min) and glucagon (retention time 36.5 min). Similar peaks were observed in control rats. The mean blood glucose variations during the primed-continuous infusion of glucagon, porcine and rat OXM are shown in Fig. 4. In phosphate buffer-infused rats, a significant decrease of the blood glucose was observed during the first 15 rain of infusion (P < 0.01), which remained stable thereafter. Whereas glucagon (1.8 nmol/kg) displayed a clear cut hyperglycemic effect throughout the infusion period, equal dosages of porcine and rat OXM induced only a small, but significant increase in blood glucose when compared with rats infused with phosphate buffer. In one set of experiments, a 10 times higher dose of rat OXM (18 nmol/kg) was infused. This produced a hyperglycemia equivalent to that measured with 1.8 nmol/kg glucagon. In this case, the mean 1000 OXM
8oo,
u
S
600,
"-~
40"
Glicentin
&)
20'
0 28
l
l
I
l
l
30
32
34
36
38
Retention time (min) Fig. 3. HPLC elution profiles of rat plasma samples, taken 45 min after the beginning of primed-continuous infusion of 1.8 nmol/kg rat OXM ( i ) or phosphate buffer (A), obtained by RIA with GOL antiserum.
48
12
3
8
_= o
4
6
15
30
45
T i m e (min) Fig. 4. Means + S.E.M. of blood glucose changes during primed-continuous infusion of isotonic phosphate buffer (x, n = 6) or of 1.8 nmol/kg of either glucagon (I-], n = 8) or porcine OXM ( A , n = 4) or rat OXM ( 0 , n = 12) and 18 nmol/kg of rat OXM ( m , n = 4). * and ***: significantly different from the corresponding values for phosphate buffer at P < 0.05 and P < 0.001, respectively.
plateau plasma concentration of GLI, as measured with GOL antiserum, was 36.7 + 1.2 pmol/ml (n = 4), which was about 10 times and 30 times higher than those obtained after infusion of 1.8 nmol/kg of rat OXM or glucagon, respectively. The plateau concentration of glucagon, as determined with GAN antiserum in the same experiment, was 517 _+ 28 fmol/ml (n = 4) and corresponded to the cross-reactivity of GAN antiserum with OXM.
Discussion
In the present, study, the in vivo plasma disappearance and metabolism of natural porcine OXM and the synthetic analog of rat OXM were compared to that ofglucagon, using primed-continuous infusion of the peptides. Under these steady-state conditions, the MCR of the peptides is independent of the number of compartments into which they are distributed and remains constant over a wide peptide concentration range [ 15,16]. The peptides were monitored by RIA, using antisera which recognized two different epitopes on each molecule; one on the central part of glucagon and OXM (GOL antiserum) [6] and the other on the C-terminal region of OXM (FAN antiserum) [14] or of glucagon (GAN antiserum) [6]. In the past, attempts to determine the metabolic fate ofglucagon-like immunoreactive materials were carried out in the dog, using a crude preparation of dog [ 17] or pig [ 18] small intestine. The estimated half-life for GLI was 20 min [ 17] and 15.9 + 1.3 min [ 18], 3 and 4 times lower, respectively, than that reported for glucagon by the same authors. However, gut GLI (enteroglucagon) does not correspond to a single molecule, which
49 limits the value of such data. Two GLI peptides have been characterized in pig small intestine: glicentin [ 19] and OXM [1,2]. In the rat, plasma enteroglucagon has been shown to correspond mainly to the association of these two molecules in a ratio of approximately 50% [6]. A single report deals with the in vivo metabolism of ~25I-glicentin in the rat [20]. The disappearance half-time of the labeled peptide, calculated from the slow (15-60 min) regression line was 59.5 + 1.8 min. However, using the fast (5-15 min) disappearance curve, a half-life close to 6 min can be estimated. In our study, both porcine and rat OXM disappeared from the plasma at a three-fold lower rate than glucagon (Table I). Similar half-disappearance times have been reported in the pig (7.2 + 0.6 min) [21] and man (8.4 + 2 min) [22] using synthetic porcine OXM. Our data indicate that substitutions of Lys by Arg (in position 33) and of Met by Nle (in position 27) in synthetic rat OXM do no modify its disappearance rate, as compared to porcine OXM. This agrees with previous observations that synthetic rat OXM displays the same biological activity as the natural porcine peptide [ 12]. The different half-lives of OXM and glucagon cannot be explained by differences in their distribution volume which are very similar and coarsely correspond to plasma and interstitial compartments. The rapid decay curves of rat OXM and glucagon in the plasma are independent of the antisera used for their monitoring since, central and C-terminal epitopes of these molecules disappeared at a similar rate (Fig. 2). However, the slow component of the disappearance curves for rat OXM differed slightly when determined by GOL and FAN antisera. This might be explained by the high specificity of FAN antiserum towards the C-terminal end of the OXM molecule [ 14], whilst GOL antiserum binds to the central region of the glucagon moiety and recognizes OXM and also the fragments of OXM bearing the epitope, which are slowly cleared from the circulation. The clearances of the peptides reflect the enzymatic cleavage at specific site(s), generating fragments which bear structurally modified epitopes. The OXM infusion (at 1.8 and 18 nmol/kg) do not increase the release of glucagon from the pancreas and no glucagon appeared to be produced, by processing of OXM to glucagon in the plasma, as demonstrated by the lack of glucagon increase measured both directly in plasma with GAN antiserum or indirectly, after HPLC, with GOL antiserum. Our results are in agreement with in vivo experiments reported in the pig [21], man [22], and with data showing that injection of ~2SI-glicentin in the rat does not generate radioactive C-terminal glucagon peptides as determined with the 30 K C-terminal-specific glucagon antiserum [20]. However, a significant transformation of gut GLI to glucagon was reported in vivo in the pig [23] and in vitro in isolated perfused pig pancreas [21], it has been reported that OXM induced a dose-dependent increase of glucagon output; this effect being observed in presence of high (15 mM) but not of physiological (5 mM) concentrations of an amino acid mixture. The MCR of glucagon, calculated from continuous infusion experiments, agrees well with that reported in therat [24,25]. It was lower than that obtained by others, using a pulse injection technique [25,26], but this difference might be partially explained by the non steady-state conditions involving uptake and diffusion of the peptide into blood and the extracellular space. The MCR of OXM was found to be 3 times slower than that of glucagon (Table I)."The~,reasons for such a difference still remain to be
50 determined. Indeed, if it is well established that glucagon is mainly cleared by the fiver and kidneys (review in Ref. 27), there have been data both supporting [28] and excluding [29] a role of the liver in the degradation of intestinal glucagon-like materials. Interestingly, it has been found that the kidney was a major site for the catabolism of glicentin, mainly through glomerular filtration and tubular catabolism [20]. In our study, acute bilateral nephrectomy reduced, by the same percentage (close to 35 ~ ) the MCR of the two peptides, which agrees with that previously reported for glucagon [24]. In sham-operated rats, infused with glucagon, the significant increase of the plateau value probably reflects the effect of operative stress, stimulating the glucagon secretion. Thus the degradation processes of the two peptides in the kidney appear to be qualitatively the same as those observed for the body as a whole. An alternative explanation for the difference in MCR of the 29- and the 37-amino acid peptides might be that the C-terminal structure of the latter is stabilized by a/~-turn locked by a salt bridge [30], whereas the C-terminal peptide of glucagon, essentially in the form of an 0c-helix [31 ], is more exposed to enzymatic breakdown by carboxypeptidases. During the course ofglucagon infusion, blood glucose was increased 2-fold while the same dose of OXM induced only a small, although significant, increase over the values in phosphate buffer-infused rats. It was necessary to increase the OXM dose 10-fold to produce the same hyperglycemia as that induced by glucagon. The hyperglycemic effect of OXM, at high concentrations, is not due to its transformation into glucagon since no C-terminal glucagon was produced during OXM infusion, as discussed above. This effect may be explained by the finding that, in vitro, OXM has been demonstrated to bind to the same receptor as that ofglucagon on rat liver membranes and to stimulate adenylate cyclase with a 5-fold and 10-fold lower potencies, respectively, than glucagon [2]. More recently, it has been reported that OXM stimulated in vitro glycogenolysis in isolated rat hepatocytes with a 10 times lower efficacity compared with glucagon [32]. Thus, the hyperglycemic effect of OXM is likely to be mediated through its binding to one or more of the glucagon receptors found in the liver [33,34].
Acknowledgements
The authors thank Mrs. Anne Cohen-Solal and Huguette Niel for their expert technical assistance. We are indebted to Dr. Jennifer Muiry for the revision of the text. This study was supported by European Economic Community (N ° 85 100001 BE 02 PUJU 1) and the Fonds pour la Recherche M6dicale.
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enteroglucagon/oxyntomodulin)from porcinejejuno-ileum. Isolation of the peptide, FEBS Lett., 146 (1982) 73-78. 2 Bataille,D., Tatemoto,K., Gespach,C. JOrnvall,H., Rosselin,G. and Mutt, V., Isolationofglucagon-37 (bioaetive enteroglucagon/oxyntomodulin)from porcinejejuno-ileum.Characterizationof the peptide, FEBS Lett., 146 (1982) 79-86.
51 3 Bell, G. I., Sanchez-Pescador, R., Laybourn, P, J. and Najarian, R. C., Exon duplication and divergence in the human preproglucagon gene, Nature, 304 (1983) 368-371. 4 Heinrich, G., Gros, P. and Habener, J. P., Glucagon gene sequence. Four of six exons encode separate functional domains of rat preproglucagon, J. Biol. Chem., 259 (1984) 14082-14087. 5 Munck A., Kervran, A., Marie, J.C., Bataille, D. and Rosselin, G., Glucagon-37 (Oxyntomodulin) and glucagon-29 (pancreatic glucagon) in human bowel: Analysis by HPLC and radioreceptorassay, Peptides, 5 (1984) 553-561. 6 Kervran, A., Blache, P. and Bataille, D., Distribution of oxyntomodulin and glucagon in the gastrointestinal tract and the plasma of the rat, Endocrinology, 121 (1987) 704-713. 7 Blache, P., Kervran, A. and Bataille, D., Oxyntomodulin and glicentin: brain-gut peptides in the rat, Endocrinology, 123 (1988) 2782-2787. 8 Dubrasquet, M., Bataille, D. and Gespach, C., Oxyntomodulin (glucagon-37 or bioactive enteroglucagon): a potent inhibitor ofpentagastrin-stimulated acid secretion in rats, Biosci. Rep., 2 (1982) 391-395. 9 Biedzinski, T.M., Bataille, D., Devaux, MA. and Sarles, H., The effect of oxyntomodulin (glucagon-37) and glucagon on exocrine pancreatic secretion in the conscious rat, Peptides, 8 (1987) 967-972. 10 Depiguy, C., Lupo, B., Kervran, A., Bataille, D., Evidence for a binding site for glucagon-37 (oxyntomodulin/bioactive enteroglucagon) in rat oxyntic glands, C. R. Acad. Sci. [D] (Paris), 299 (1984) 677-680. 11 Seal, A., Yamada, T., Debas, H., Hollinshead, J., Osadchey, B., Aponte, G. and Walsh, J., Somatostatin-14 and -28: clearance and potency on gastric function in dogs, Amer. J. Physiol. 243 (1982) G97-GI02. 12 Audousset-Puech, M.P., Dufour, M., Kervran, A., Jarrousse, C., Castro, B., Bataille, D. and Martinez, J. Solid-phase peptide synthesis of human (Nle-27)-oxyntomodulin. Preliminary evaluation of this biological activities, FEBS Lett., 200 (1986)181-185. 13 Blache, P., Kervran, A., Dufour, M. and Bataille, D., Preparation, purification and characterization of iodoglucagons, Regul. Pept., $3 (1985) A 20. 14 Blache, P., Kervran, A., Martinez, J. and Bataille, D., Development of an oxyntomodulin/glicentin C-terminal radioimmunoassay using a 'thiol-maleoyr coupling method for preparing the immunogen, Anal. Biochem., 173 (1988) 171-179. 15 Tait, P.G., Review: The use of isotopic steroids for measurement of production rates in vivo, J. Clin. Endocrinol. 23 (1963) 1285-1297. 16 Goldstein, A., Aronow, L., Kalman, S.M., Principles of drug action. The basis of pharmacology, Harper & Row, New York 1969, 292 pp. 17 Unger, R. H., Ohneda, A., Valverde,I., Eisentraut, A. M. and Exton, J., Characterization ofthe responses of circulating glucagon-like immunoreactivity to intraduodenal and intravenous administration of glucose, J. Clin. Invest., 47 (1968) 48-65. 18 Tanaka, R., Matsuyama, T., Shima, K., Sawazaki, N., Tarui, S. and Kumahara, Y., Half life of gastrointestinal glucagon-like immunoreactive materials, Endocrinol. Jpn., 26 (1979) 59-63. 19 Thim, L. and Moody, A.J., The primary structure of porcine glicentin (proglucagon), Regul. Pept., 2 (1981 ) 139-150. 20 Lopez-Novoa, J.M., Santos, J.C., Villamediana, L.M., Garrote, F.J., Thim, L., Moody, A.J. and Valverde, I. Renal catabolism of 12SI-glicentin, Amer. J. Physiol., 250 (1986) E545-E550. 21 Baldissera, F.G.A., Hoist, J.J., Knuhtsen, S., Hilsted, L. and Nielsen, O.V., Oxyntomodulin (glicentin(33-69)): pharmacokinetics, binding to liver cell membranes, effects on isolated perfused pig pancreas, and secretion from isolated perfused lower small intestine of pigs, Regul. Pept., 21 (1988) 151-166. 22 Schjoldager, B.,Mortensen, P.E.,Myhre, J.,Christiansen, J. and Holst, J.J.,Oxyntomodulin from distal gut: Role in regulation of gastric and pancreatic functions, Dig. Dis. Sci. 34 (1989) 1411-1419. 23 Koranyi, L.,Peterfy, F., Szabo,J.,TSrSk, A.,Guoth, M. andTamas, Jr., G., Evidence for transformation of glucagon-like immunoreactivity of gut into pancreatic glucagon in vivo, Diabetes, 30 (1981) 792-794. 24 Emmanouel, D. S., Jaspan, J.B., Rubenstein, A.H., Huen, A. H.J., Fink, E. and Katz, A.I., Glucagon metabolism in the rat. Contribution of the kidney to the metabolic clearance rate of the hormone,J. Clin. Invest., 61 (1978) 6-13. 25 Oshima, I., Hirota, M., Ohboshi, C. and Shima, K., Comparison of half-disappearance times, distribu-
52 tion volumes and metabolic clearance rates of exogenous glucagon-like peptide 1 and glucagon in rats, Regul. Pept., 21 (1988) 85-93. 26 Balage, M. and Grizard, J., Glucagon kinetics in growing rats fed different levels of protein and/or energy, Reprod. Nutr. D6velop., 24 (1984) 251-263. 27 Polonsky, K. S., Jaspan, J.B. and Rubenstein, A.H., The metabolic clearance rate of glucagon. In P.J. Lefebvre (Ed.), Glucagon II, Springer-Verlag, Berlin, 1983, pp. 353-359. 28 Buchanan, K. D., Solomon, S. S., Vance, J. E., Porter, H.P. and Williams, R. H., Glucagon clearance by the isolated perfused rat liver, Proc. Soc. Exp. Biol. Med., 128 (1968) 620-623. 29 Gutman, R. A., Fink, G., Voyles, N., Selawry, H., Penhos, J. C., Lepp, A. and Recant, L., Specific biologic effects of intestinal glucagon-like materials, J. Clin. Invest., 52 (1973) 1165-1175. 30 Aumelas A., Audousset-Puech, M. P., Heitz, A., Bataille, D. and Martinez, J., H n.m.r, contbrmational studies on the C-terminal octapeptide of oxyntomodulin, a fl-turn locked by a salt bridge, Int. J. Peptide Protein Res., 34 (1989) 268-276. 31 Braun, W., Wider, G., Lee, K.H., and WOthrick, K., Conformation of glucagon in a lipid-water interphase by 1H nuclear magnetic resonance, J. Mol. Biol., 169 (1983) 921-948. 32 Kojima, H., Suzuki, M., Hidaka, H., Yanaihara, N., Yanaihara, C. and Harano, Y., Biological activity of oxyntomodulin in hepatocytes, Biomed. Res., 9 ( 1988) 191-194. 33 Wakelam, M. J. O., Murphy, G. J., Hruby, V.J. and Houslay, M. D., Activation of two signal-transduction systems in hepatocytes by glucagon, Nature, 323 (1986) 68-71. 34 Bataille, D., Blache, P., Mercier, F., Jarrousse, C., Kervran, A., Dufour, M., Mangeat, P., Dubrasquet, M., Mallat, A., Lotersztajn, S., Pavoine, C. and Pecker, F., Glucagon and related peptides: Molecular structure and biological specificity, Ann. NY Acad. Sci., 527 (1988) 168-185.
41
REGPEP 00964
Metabolic clearance rates of oxyntomodulin and glucagon in the rat: contribution of the kidney A. Kervran ~.t, M. Dubrasquet 1,,, p. Blache ~, J. Martinez ~ and D. B ataille I Centre CNRS-INSERM de Pharmacologie-Endocrinologie, Montpellier and 21NSERM U. 10, Hrpital Bichat, Paris (France) (Received 10 January 1990; revised version received 16 July 1990; accepted 18 July 1990)
Key words: Half-life; Primed-continuous infusion technique; Nephrectomy; Hyperglycemic effect
Summary The half-life (t,/:) and metabolic clearance rate (MCR) of exogenous natural porcine oxyntomodulin (porcine OXM) and the synthetic analog of rat oxyntomodulin, [NIe27]-OXM (rat OXM), were compared with that of glucagon in control, shamoperated and acutely nephrectomized rats using the primed-continuous infusion technique. The half-disappearance times for porcine OXM (8.2 + 0.5 min) and rat OXM (6.4 + 0.5 min) were 3-fold slower than that ofglucagon (1.9 + 0.1 min). Acute bilateral nephrectomy significantly prolonged the half-disappearance time of rat OXM (8.2 + 0.7 min) and glucagon (3.6 + 0.4 min) compared with that of sham-operated animals (6.5 + 0.8 min and 2.5 + 0.2 min, respectively). The mean MCRs were similar for porcine and rat OXM (11.3 + 0.7 and 11.9 + 0.5 ml- kg- 1. min - 1) but were 3 times lower than that measured with glucagon (36 + 5 ml. kg- 1. min- 1). Bilateral nephrectomy reduced the MCR of OXM and glucagon by 38 Yo and 34Yo, respectively. No significant increase in C-terminal glucagon immunoreactivity was noticed during infusion of either porcine or rat OXM, measured directly in plasma, with a specific C-terminal glucagon antiserum or after HPLC. In the course of the glucagon infusion, blood glucose was increased 2-fold, while the same dose of porcine OXM or of rat OXM induced only a small increase over the values in phosphate buffer-infused rats. 10 times higher doses of rat OXM were necessary to obtain a similar hyperglycemic effect. These results indicate that: (1) the metabolism of OXM is 3-fold slower than that of * Deceased in 1990. Correspondence: A. Kervran, Centre CNRS-INSERM de Pharmacologie-Endocrinologie, 34094 Montpellier Cedex 5, France. 0167-0115/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
42 glucagon, (2) renal clearance contributed close to 35 ~o of the overall metabolic plasma extraction for OXM and glucagon and (3) OXM, although effective at a higher dose, when compared with glucagon, displays a hyperglycemic effect probably through the glucagon receptors.
Introduction
Oxyntomodulin (OXM) is a glucagon-containing peptide, initially isolated from porcine jejuno-ileum [1], which contains the sequence of glucagon, extended at its C-terminal end by a basic octapeptide [2]. The primary structure of human and rat OXM, deduced from the nucleotide sequence of their cDNAs are identical [3,4] and differs from that of porcine OXM by a single amino acid (Arg vs. Lys) in position 33. The distribution of OXM has been determined in the human and rat bowel [5,6] and in the rat central nervous system [7]. In rat plasma, the OXM concentration increased 2-fold 2 h after a solid meal [6]. The basic octapeptide which differentiates OXM from glucagon, confers biological activities which are quantitatively different: in vivo, OXM is 15-20 times more potent than glucagon in inhibiting pentagastrin-induced gastric acid secretion in the rat [8]; it is also one order of magnitude more potent than glucagon in inhibiting exocrine pancreatic secretion [9]. In vitro, binding sites in oxyntic gland prepared from rat gastric mucosa [ 10] had affinities 10 times higher for OXM than for glucagon. In contrast, OXM is 10 times less effective than glucagon for the production of cAMP in rat liver membranes [2]. Differences in the plasma half-life of peptides existing in different forms and bearing the same biological activity (as well as their potential interconversion from one form to another) may alter substantially their relative potencies in in vivo experiments performed on a molar-dose basis. Indeed, this was the case for somatostatin-14 and -28 [11]. Therefore, our study was designed: (1) to compare the disappearance kinetics of natural porcine OXM and synthetic analog of rat OXM to that of glucagon, (2) to determine whether the metabolism of OXM results in a significant conversion to glucagon and (3) to investigate the contribution of the kidney in the handling of these peptides.
Materials and Methods
Peptide infusion procedure and samples Male Wistar rats (250-280 g) fed ad libitum, were anesthetized by an intraperitoneal injection of pentobarbital (30 mg/kg body weight). Peptide challenging was carried out 30 min later using a primed-continuous infusion of 1.8 nmol/kg/45 min, in isotonic phosphate buffer (pH 7.4), through the saphenous vein: the prime dose (0.45 nmol/kg) was administered over 2 rain at a flow rate of 0.5 ml/min and the continuous infusion, over a period of 43 min, at a flow rate of 70/~l/min. This dosage was chosen because, in pilot experiments, it was found to induce a steady state of peptide concentration
43 within the first 15 rain of infusion. In a set of experiments, rats were bilaterally nephrectomized or sham-operated 30 min before peptide challenge. Blood samples of 0.5 ml were taken from a carotid catheter at 0, 15, 30 and 45 min throughout infusion and, at the end of infusion, at 1, 2.5, 5, 7.5, 10 and 20 min. Blood samples were collected in chilled tubes containing antiprotease (500 KIU/ml aprotinin, Biosys, Compirgne, France) and EDTA (1 mg/ml), centrifuged at 4 °C and stored at 20 ° C until assay. -
Peptides and chemicals Highly purified porcine OXM, used for infusion and column calibration, was obtained as previously described [1,2]. The synthetic analog of rat OXM [NleZT]-OXM) was obtained by solid-phase synthesis, achieved in our laboratory [12]. Rat glicentin was partially purified on a Bio-Gel P 10 column (Bio-Rad, Richmond, CA) and by reversedphase HPLC as previously described [6]. Highly purified porcine pancreatic glucagon was purchased from the Novo Research Institute (Bagsvaerd, Denmark). All solvents and reagents were of analytical grade. Reversed-phase HPLC Glucagon-containing peptides were quantitatively extracted from 5 ml plasma samples on Sep-Pak cartridges (Waters Associates, Millipore, Velizy, France) as previously described [6]. Eluates were separated at room temperature by reversed-phase HPLC on two #Bondapak C~8 columns (i.d. 0.39 x 30 cm, Waters) in series and a C18 precolumn (Waters), using a Waters chromatograph composed of two model 6000 A pumps, and a model 720 solvent programmer, a Rheodyne (model 7010) sample injector (Touzart et Matignon, Paris, France), fitted with an 18-ml loop. Reversed-phase chromatography were performed, as reported elsewhere [6] with a 45-min linear gradient of 20-50~o acetonitrile (SDS, Peypin, France) in aqueous solvent consisting of 1~o trifluoroacetic acid (Fluka, Buchs, Switzerland) buffered to pH 2.5 by diethylamine (J. T. Baker Chemicals, Deventer, The Netherlands), at a flow rate of 1.5 ml/min. Fractions were collected every 0.3 min. Chemical and immunological analyses Blood glucose concentration was measured using a glucose-oxidase method (Biochemica test combination, Boehringer, Mannheim, F.R.G.). Glucagon and glucagon-like immunoreactivity (GLI) were determined by RIAs using the C-terminal glucagon antiserum GAN and the centrally-directed glucagon antiserum GOL, as previously reported [6]. GOL antiserum displayed a full cross-reactivity with glucagon, OXM and partially purified rat glicentin and GAN antiserum showed a 2~o cross-reaction with OXM [6]. Mono 12SI-glucagon [ 13] was used as tracer and the separation of free from bound peptides was performed by adsorption on dextran-coated charcoal. Peptides bearing the C-terminal octapeptide (glicentin and OXM) were measured with an OXM/glicentin C-terminal RIA using FAN antiserum as previously reported [ 14]. In brief, the C-terminal nonadecapeptide (OXM 19-37), which fully cross-reacted with OXM and partially purified rat glicentin, and mono [ 125I-Tyr]-OXM 19-37 were used as standard and as tracer, respectively. Separation of free from bound peptides
44 was obtained by adsorption on dextran-coated charcoal. The sensitivity of FAN antiserum was 2 fmol/tube and the intra- and inter-assay reproducibilities at this concentration were 13 + 2~o (n = 16) and 23 + 3~o (n = 6), respectively.
Calculations Glucagon and OXM data, obtained from infusion studies, were analyzed according to the plateau principle [ 15,16]. Plasma steady state concentrations of glucagon or OXM were taken as the mean of the three values obtained at 15, 30 and 45 min. At the end of peptide infusion, the increment of glucagon and GLI concentrations, above the preinfusion levels, were expressed as percent of the mean plateau value, plotted on a logarithmic scale versus time and computed to yield the slope from which the tl/2 was determined. The metabolic clearance rate (MCR), expressed in ml. kg- 1. min - ~ was calculated by dividing the infused dose (pmol • kg- ~• min- 1) by the plateau increment in plasma peptide (pmol- ml- 1). The apparent volume of distribution (in ml. k g - 1) was obtained by dividing the MCR by the slope of the regression line for disappearance. Linear regression analysis was determined by the least-squares method. All values shown represent the mean + S.E.M. Differences between means was evaluated by paired or unpaired Student's t-test, as required. In this study we assumed that endogenous production of glucagon or of OXM remained constant during the exogenous infusion experiments.This assumption is indirectly supported by the steady level of glucagon, as determined by GAN antiserum, during OXM infusion (Fig. lb and c).
Results
The time-courses of the mean plasma concentrations ofglucagon and GLI during the intravenous primed-continuous peptide infusion were measured by the C-terminal glucagon antiserum (GAN) and the centrally-directed glucagon antiserum (GOL), respectively, and are shown Fig. 1. The preinfusion values of glucagon and GLI, expressed in fmol/ml of plasma glucagon equivalent, were (n = 24) 34 + 2 and 105 + 4, respectively. Plateau values were observed between 15 and 45 min after the start of the infusion of 1.8 nmol/kg of the peptides. The coefficients of variation at 15-45'min, during glucagon infusion (Fig. la), porcine OXM infusion (Fig. lb), and rat OXM infusion(Fig, lc),were6 + 1 ~ , 8 + 3~o and 10 + l~o,respectively. Theplateau values, during porcine or rat OXM infusions (Table I), were about 2.5-fold higher than that observed during glucagon infusion and they were significantly increased in nephrectomized animals (P < 0.05). During the course of infusions of porcine and rat OXM, the plateau values for glucagon, as measured with GAN antiserum, were 83 + 9 fmol/ml and 70 + 9 fmol/ml, respectively, and were significantly higher than the preinfusion level (P < 0.001). This increment, over baseline values, corresponded to 1-1.5 ~o of the plasma GLI concentration, as determined with GOL antiserum and was in good agreement with the 2~o cross-reactivity of GAN antiserum with OXM (see Materials and Methods). Thus,
45 .
a = GOL antiserum • GAN ~mfise~m
~
2'
r. 0
o
1~ T
3b
i m e
-
~
4~
60
0
~
3~ T i m e
(rain)
4~
6~
(min)
4
} e~
g2 0 OlD
E
r. o
1~
ab
4;
T i m e
(rain)
6b
Fig. 1. Plasma concentrations of C-terminal ( m ) and central (r-q) glucagon-like immunoreactivity during and after 45 min intravenous primed-continuous infusion of 1.8 nmol/kg of: (a) glucagon (n = 8), (b) porcine O X M (n = 4) and (c)rat O X M (n = 12). Symbols and bars are m e a n s _+ S.E.M.
changes in G L I concentrations during infusion of porcine or of rat O X M can be attributed to an increase in these peptides and not to a stimulatory effect on glucagon secretion. When the peptide infusion was discontinued, circulating glucagon and G L I disappeared within the 20-min period studied, according to bi-exponential curves which are indicated on a semilogarithmic scale in Fig. 2. Examination of the decay regression
46 TABLE I Comparison of plasma half-lives (t~a), metabolic clearance rate (MCR) and apparent distribution volume (Vd) for glucagon, natural porcine oxyntomodulin (pOXM) and synthetic rat oxyntomodulin (rOXM) in control, sham-operated and nephrectomized rats Animals
Peptides
n
Plateau value (fmol/ml)
t~a (min)
MCR (ml. k g - i. min - ])
Vd (ml. kg - ~)
Control
Glucagon pOXM rOXM Glucagon rOXM Glucagon rOXM
8 4 12 5 5 5 5
1302 3669 3532 1715 3214 2095 5312
1.9 8.2 6.4 2.5 6.5 3.6 8.2
36.4 11.3 11.9 29.2 13.0 19.4 8.0
101 135 112 106 122 100 95
Sham-operated Nephrectomized
+ 104 _+ 206 + 149 + 53 + 64 _+ 153 + 257
+ 0.1 + 0.5 + 0.5 + 0.2 _+ 0.4 _+ 0.4 + 0.5
+ + + + + + +
4.5 0.7 0.5 1.0 0.3 1.0 0.2
+ 16 _+ 13 + 12 + 9 + 18 + 13 + 8
Values shown are the mean + S.E.M.
lines revealed that the disappearance rate of glucagon was much faster than that of porcine or rat OXM. The calculated values for plasma half-lives, MCR and apparent volume of distribution from the rapid decay regression lines, obtained with GOL antiserum, are provided in Table I. The t, n for porcine and rat OXM, as measured with GOL were not significantly different (P > 0.05) for the fast (Table I) and the slow (28.2 + 5.4 min vs. 23.8 + 3.5 min, respectively) regression lines. The t,/2 for glucagon 100
~2 10
1'0
1'5
Time (min) Fig. 2. Disappearance rates o f p o r c i n e O X M ( , ) , r a t O X M (F], O ) and glucagon ( A , Z~) from the plasma, as measured with G O L antiserum ( m , F], A ) , G A N antiserum ( A ) and F A N antiserum ( 0 ) . Peptide concentrations are expressed as percent of the mean 45 min plateau value. Results are m e a n s _+ S.E.M.
47
and rat OXM, were not statistically different whatever the region specific antisera used (Fig. 2). However, a significant difference was seen for the slow component of the decay regression line of rat OXM, when measured with FAN antiserum (12.8 + 1.6 min, P < 0.05). Infusion of glucagon in sham-operated rats elicited a significant increase of the plateau value (P < 0.01) and plasma t,/~ (P < 0.02), as compared to the control rats. Such an effect was not observed in sham-operated rats infused with rat OXM. Acute bilateral nephrectomy significantly prolonged the plasma half-lives of glucagon and rat OXM (P < 0.05) and decreased their MCR (P < 0.001) by 34% and 38~o, respectively, as compared to the sham-operated values. The reversed-phase HPLC profile of plasma samples (Fig. 3), taken 45 min after the start of rat OXM infusion (1.8 nmol/kg/45 min), revealed an increase in a single major peak of activity, when compared with a control plasma elution profile, that eluted in the position of the pure rat OXM peptide. Two small peaks were also seen on the elution profiles, corresponding to glicentin (retention time 30.5 min) and glucagon (retention time 36.5 min). Similar peaks were observed in control rats. The mean blood glucose variations during the primed-continuous infusion of glucagon, porcine and rat OXM are shown in Fig. 4. In phosphate buffer-infused rats, a significant decrease of the blood glucose was observed during the first 15 rain of infusion (P < 0.01), which remained stable thereafter. Whereas glucagon (1.8 nmol/kg) displayed a clear cut hyperglycemic effect throughout the infusion period, equal dosages of porcine and rat OXM induced only a small, but significant increase in blood glucose when compared with rats infused with phosphate buffer. In one set of experiments, a 10 times higher dose of rat OXM (18 nmol/kg) was infused. This produced a hyperglycemia equivalent to that measured with 1.8 nmol/kg glucagon. In this case, the mean 1000 OXM
8oo,
u
S
600,
"-~
40"
Glicentin
&)
20'
0 28
l
l
I
l
l
30
32
34
36
38
Retention time (min) Fig. 3. HPLC elution profiles of rat plasma samples, taken 45 min after the beginning of primed-continuous infusion of 1.8 nmol/kg rat OXM ( i ) or phosphate buffer (A), obtained by RIA with GOL antiserum.
48
12
3
8
_= o
4
6
15
30
45
T i m e (min) Fig. 4. Means + S.E.M. of blood glucose changes during primed-continuous infusion of isotonic phosphate buffer (x, n = 6) or of 1.8 nmol/kg of either glucagon (I-], n = 8) or porcine OXM ( A , n = 4) or rat OXM ( 0 , n = 12) and 18 nmol/kg of rat OXM ( m , n = 4). * and ***: significantly different from the corresponding values for phosphate buffer at P < 0.05 and P < 0.001, respectively.
plateau plasma concentration of GLI, as measured with GOL antiserum, was 36.7 + 1.2 pmol/ml (n = 4), which was about 10 times and 30 times higher than those obtained after infusion of 1.8 nmol/kg of rat OXM or glucagon, respectively. The plateau concentration of glucagon, as determined with GAN antiserum in the same experiment, was 517 _+ 28 fmol/ml (n = 4) and corresponded to the cross-reactivity of GAN antiserum with OXM.
Discussion
In the present, study, the in vivo plasma disappearance and metabolism of natural porcine OXM and the synthetic analog of rat OXM were compared to that ofglucagon, using primed-continuous infusion of the peptides. Under these steady-state conditions, the MCR of the peptides is independent of the number of compartments into which they are distributed and remains constant over a wide peptide concentration range [ 15,16]. The peptides were monitored by RIA, using antisera which recognized two different epitopes on each molecule; one on the central part of glucagon and OXM (GOL antiserum) [6] and the other on the C-terminal region of OXM (FAN antiserum) [14] or of glucagon (GAN antiserum) [6]. In the past, attempts to determine the metabolic fate ofglucagon-like immunoreactive materials were carried out in the dog, using a crude preparation of dog [ 17] or pig [ 18] small intestine. The estimated half-life for GLI was 20 min [ 17] and 15.9 + 1.3 min [ 18], 3 and 4 times lower, respectively, than that reported for glucagon by the same authors. However, gut GLI (enteroglucagon) does not correspond to a single molecule, which
49 limits the value of such data. Two GLI peptides have been characterized in pig small intestine: glicentin [ 19] and OXM [1,2]. In the rat, plasma enteroglucagon has been shown to correspond mainly to the association of these two molecules in a ratio of approximately 50% [6]. A single report deals with the in vivo metabolism of ~25I-glicentin in the rat [20]. The disappearance half-time of the labeled peptide, calculated from the slow (15-60 min) regression line was 59.5 + 1.8 min. However, using the fast (5-15 min) disappearance curve, a half-life close to 6 min can be estimated. In our study, both porcine and rat OXM disappeared from the plasma at a three-fold lower rate than glucagon (Table I). Similar half-disappearance times have been reported in the pig (7.2 + 0.6 min) [21] and man (8.4 + 2 min) [22] using synthetic porcine OXM. Our data indicate that substitutions of Lys by Arg (in position 33) and of Met by Nle (in position 27) in synthetic rat OXM do no modify its disappearance rate, as compared to porcine OXM. This agrees with previous observations that synthetic rat OXM displays the same biological activity as the natural porcine peptide [ 12]. The different half-lives of OXM and glucagon cannot be explained by differences in their distribution volume which are very similar and coarsely correspond to plasma and interstitial compartments. The rapid decay curves of rat OXM and glucagon in the plasma are independent of the antisera used for their monitoring since, central and C-terminal epitopes of these molecules disappeared at a similar rate (Fig. 2). However, the slow component of the disappearance curves for rat OXM differed slightly when determined by GOL and FAN antisera. This might be explained by the high specificity of FAN antiserum towards the C-terminal end of the OXM molecule [ 14], whilst GOL antiserum binds to the central region of the glucagon moiety and recognizes OXM and also the fragments of OXM bearing the epitope, which are slowly cleared from the circulation. The clearances of the peptides reflect the enzymatic cleavage at specific site(s), generating fragments which bear structurally modified epitopes. The OXM infusion (at 1.8 and 18 nmol/kg) do not increase the release of glucagon from the pancreas and no glucagon appeared to be produced, by processing of OXM to glucagon in the plasma, as demonstrated by the lack of glucagon increase measured both directly in plasma with GAN antiserum or indirectly, after HPLC, with GOL antiserum. Our results are in agreement with in vivo experiments reported in the pig [21], man [22], and with data showing that injection of ~2SI-glicentin in the rat does not generate radioactive C-terminal glucagon peptides as determined with the 30 K C-terminal-specific glucagon antiserum [20]. However, a significant transformation of gut GLI to glucagon was reported in vivo in the pig [23] and in vitro in isolated perfused pig pancreas [21], it has been reported that OXM induced a dose-dependent increase of glucagon output; this effect being observed in presence of high (15 mM) but not of physiological (5 mM) concentrations of an amino acid mixture. The MCR of glucagon, calculated from continuous infusion experiments, agrees well with that reported in therat [24,25]. It was lower than that obtained by others, using a pulse injection technique [25,26], but this difference might be partially explained by the non steady-state conditions involving uptake and diffusion of the peptide into blood and the extracellular space. The MCR of OXM was found to be 3 times slower than that of glucagon (Table I)."The~,reasons for such a difference still remain to be
50 determined. Indeed, if it is well established that glucagon is mainly cleared by the fiver and kidneys (review in Ref. 27), there have been data both supporting [28] and excluding [29] a role of the liver in the degradation of intestinal glucagon-like materials. Interestingly, it has been found that the kidney was a major site for the catabolism of glicentin, mainly through glomerular filtration and tubular catabolism [20]. In our study, acute bilateral nephrectomy reduced, by the same percentage (close to 35 ~ ) the MCR of the two peptides, which agrees with that previously reported for glucagon [24]. In sham-operated rats, infused with glucagon, the significant increase of the plateau value probably reflects the effect of operative stress, stimulating the glucagon secretion. Thus the degradation processes of the two peptides in the kidney appear to be qualitatively the same as those observed for the body as a whole. An alternative explanation for the difference in MCR of the 29- and the 37-amino acid peptides might be that the C-terminal structure of the latter is stabilized by a/~-turn locked by a salt bridge [30], whereas the C-terminal peptide of glucagon, essentially in the form of an 0c-helix [31 ], is more exposed to enzymatic breakdown by carboxypeptidases. During the course ofglucagon infusion, blood glucose was increased 2-fold while the same dose of OXM induced only a small, although significant, increase over the values in phosphate buffer-infused rats. It was necessary to increase the OXM dose 10-fold to produce the same hyperglycemia as that induced by glucagon. The hyperglycemic effect of OXM, at high concentrations, is not due to its transformation into glucagon since no C-terminal glucagon was produced during OXM infusion, as discussed above. This effect may be explained by the finding that, in vitro, OXM has been demonstrated to bind to the same receptor as that ofglucagon on rat liver membranes and to stimulate adenylate cyclase with a 5-fold and 10-fold lower potencies, respectively, than glucagon [2]. More recently, it has been reported that OXM stimulated in vitro glycogenolysis in isolated rat hepatocytes with a 10 times lower efficacity compared with glucagon [32]. Thus, the hyperglycemic effect of OXM is likely to be mediated through its binding to one or more of the glucagon receptors found in the liver [33,34].
Acknowledgements
The authors thank Mrs. Anne Cohen-Solal and Huguette Niel for their expert technical assistance. We are indebted to Dr. Jennifer Muiry for the revision of the text. This study was supported by European Economic Community (N ° 85 100001 BE 02 PUJU 1) and the Fonds pour la Recherche M6dicale.
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